The present invention relates to the field of plant biotechnology. More specifically, it concerns methods of incorporating genetic components into the genome of monocotyledonous or dicotyledonous plants. In particular, provided herein are reproducible systems for genetically transforming corn, soybean, rice and wheat. Most particularly, there is a system for transforming wheat.
The method comprises novel conditions during co-culture of Agrobacterium with a regenerable plant cell or tissue. Exemplary methods include an improved method using Agrobacterium-mediated transformation for introducing nucleic acids into different regenerable tissues using a variety of selectable or screenable marker systems, and with a number of different plant species. The present invention also provides fertile transgenic plants, particularly wheat. In other aspects, the invention relates to the production of stable transformed and fertile plants, gametes, and offspring from these plants.
During the past decade, it has become possible to transfer genes from a wide range of organisms to crop plants by recombinant DNA technology. This advance has provided enormous opportunities to improve plant resistance to pests, disease and herbicides, and to modify biosynthetic processes to change the quality of plant products (Knutson et al., 1992; Piorer et al., 1992; Vasil et al., 1992). However, the availability of efficient Agrobacterium-mediated transformation methods suitable for high capacity production of economically important plants is limited.
There have been many methods attempted for plant transformation, but only a few methods are highly efficient. Methods for DNA transformation of plant cells include Agrobacterium-mediated plant transformation (see, for example, U.S. Pat. Nos. 5,416,011 and 5,569,834 and WO 97/48814). In addition, protoplast transformation, gene transfer into pollen, injection into reproductive organs, injection into immature embryos, and particle bombardment have been employed for plant transformation. Despite the number of transformation methods available for specific plant systems, it would be advantageous to have one method of introducing genes into plants that is applicable to several different crops and a variety of regenerable tissues.
Several technologies for the introduction of DNA into cells are well known to those of skill in the art and can be divided into categories including: (1) chemical methods (Graham and van der Eb, 1973; Zatloukal et al., 1992); (2) physical methods such as microinjection (Capecchi, 1980), electroporation (Wong and Neuman, 1982; Fromm et al., 1985; U.S. Pat. No. 5,384,253), and the gene gun (Johnston and Tang, 1994; Fynan et al., 1993); (3) viral vectors (Clapp, 1993; Lu et al., 1993; Eglitis and Anderson, 1988); and (4) receptor-mediated mechanisms (Curiel et al., 1992; Wagner et al., 1992);
Until recently, the methods employed for some monocot species included direct DNA transfer into isolated protoplasts and microprojectile-mediated DNA delivery (Shimamoto et al., 1989; Fromm et al, 1990). The protoplast methods have been widely used in rice, where DNA is delivered to the protoplasts through liposomes, PEG, and electroporation. While a large number of transgenic plants have been recovered in several laboratories (Shimamoto et al., 1989; Datta et al., 1990), the protoplast methods require the establishment of long-term embryogenic suspension cultures. Some regenerants from protoplasts are infertile and phenotypically abnormal due to the long-term suspension culture (Davey et al., 1991; Rhodes et al.,1988). U.S. Pat. No. 5,631,152 describes a rapid and efficient microprojectile bombardment method for the transformation and regeneration of monocots and dicots.
More recently, monocot species have been successfully transformed via Agrobacterium-mediated transformation. WO 97/48814 discloses processes for producing stably transformed fertile wheat. The method describes the recovery of transgenic, wheat plants within a short period of time using a variety of explants. Agrobacterium-mediated transformation provides a viable alternative to bombardment methods, and the method also allows quick molecular analysis of transgenic lines.
The major deficiencies in current plant transformation systems utilizing Agrobacterium-mediated methods include the production efficiency of the system and transformation difficulties due to genotype or species diversity and explant limitations. WO 94/00977 describes a method for transforming monocots that depends on the use of freshly cultured immature embryos for one monocot and cultured immature embryos or callus for a different monocot. In either system, the explants must be freshly isolated, and the method is labor intensive, genotype-, and explant-limited. Other reports rely on the use of specific strains or vectors to achieve high efficiency transformation. In one report, a specific super binary vector must be used in order to achieve high-efficiency transformation (Ishida et al., 1996).
Despite the number of transformation methods in the art, few methods have been developed that are applicable to both monocots and dicots. The present invention provides an improvement of an Agrobacterium-mediated transformation method. The method is more efficient in delivering target DNA to the plant as evidenced by higher transformation efficiencies and provides a reduction in labor and cost advantage compared with conventional methods.